SP-A、SP-D在茄病镰刀菌感染大鼠角膜组织中早期表达的实验研究

发布时间：2018-08-24 13:41

【摘要】： 目的：检测茄病镰刀菌感染大鼠角膜早期表面活性蛋白A和D (surfactant protein A and D,SPA、SP-D)的表达,初步探讨SP-A、SP-D在真菌性角膜炎固有免疫中的作用。方法：Wistar大鼠80只随机分成4组：空白对照组、单纯损伤组、真菌性角膜炎一组、真菌性角膜炎二组。右眼为实验眼。空白对照组5只大鼠,不做任何处理；单纯损伤组25只模拟模型的制作不接种茄病镰刀菌；真菌性角膜炎一组、真菌性角膜炎二组各25只建立茄病镰刀菌性角膜炎动物模型。单纯损伤组、真菌性角膜炎一组、真菌性角膜炎二组分别于建模后6h、12h、24h、48h、96h处死5只大鼠。各组分别在裂隙灯显微镜下观察大鼠的眼部变化；病理组织切片观察角膜组织炎症程度；(空白对照组、单纯损伤组、真菌性角膜炎一组取右眼角膜,真菌性角膜炎二组取左眼角膜)逆转录聚合酶链式反应(RT-PCR)检测角膜组织SP-A、SP-D mRNA表达；免疫组化技术检测角膜组织中SP-A、SP-D的蛋白表达以及共表达强度。结果：(1)裂隙灯检查：真菌感染角膜6h与单纯损伤组比较大鼠眼部没有明显差异,睑球结膜充血(+),角膜水肿不明显；12h睑球结膜充血(++),角膜出现水肿；24h睑球结膜充血(+++),角膜毛玻璃样水肿；48h、96h角膜水肿明显,出现黄白色溃疡,溃疡面积大小不等,免疫环形成。(2)病理组织学检查：真菌感染角膜6h与单纯损伤组比较没有明显差异,角膜中心上皮层缺如,胶原纤维排列整齐,未见明显中性粒细胞浸润；12h基质浅层少量中性粒细胞侵入；24h角膜水肿,角膜中心上皮和前弹力层缺失,基质层胶原纤维排列紊乱,基质浅层中性粒细胞侵润增加；48h、96h角膜水肿明显,角膜基质排列更加紊乱,基质全层大量中性粒细胞浸润。(3)免疫组化检测：正常大鼠、单纯损伤组以及真菌性角膜炎二组角膜上皮细胞有微弱的SP-A蛋白、SP-D蛋白的表达,三组比较没有明显差异(p0.05)；真菌感染角膜6h角膜上皮细胞SP-A蛋白及SP-D蛋白表达没有明显增强,与正常角膜、单纯损伤组以及真菌性角膜炎二组比较没有明显差异(p0.05),12h后表达逐渐增强,48h最强,96h下降,各时间点总体比较表达水平差异有显著性(p0.01),与空白对照组以及单纯损伤组和真菌性角膜炎二组相同时间点比较表达水平差异有显著性(p0.01)。(4) RT-PCR检测：正常大鼠、单纯损伤组、真菌性角膜炎二组角膜有少量SP-A mRNA、SP-D mRNA的表达,三组比较没有明显差异(p0.05)；真菌感染角膜后SP-A mRNA、SP-D mRNA的表达量逐渐增加,24h最高,48h、96h下降,各时间点总体比较表达水平差异有显著性(p0.01),与空白对照组以及单纯损伤组和真菌性角膜炎二组相同时间点比较具有显著差异(p0.01) 结论：茄病镰刀菌感染角膜的早期SP-A、SP-D在角膜组织表达显著增加,随着病情进展表达有所下降,但仍高于单纯损伤组。提示SP-A、SP-D可能为茄病镰刀菌的模式识别受体,在角膜抵抗茄病镰刀菌感染早期固有免疫应答中发挥着一定作用。
[Abstract]:Objective: To detect the expression of surfactant protein A and D (SPA, SP-D) in the cornea of rats infected with Fusarium solanacearum at early stage, and to explore the role of SP-A and SP-D in the innate immunity of fungal keratitis.
Methods: 80 Wistar rats were randomly divided into 4 groups: blank control group, simple injury group, fungal keratitis group, fungal keratitis group, right eye, blank control group, 5 rats without any treatment, simple injury group, 25 model rats without inoculation of Fusarium solani, fungal keratitis group, fungal cornea group. Five rats were sacrificed at 6 h, 12 h, 24 h, 48 h and 96 h after the models were established in the simple injury group, fungal keratitis group and fungal keratitis group. (blank control group, simple injury group, fungal keratitis group take right eye cornea, fungal keratitis group take left eye cornea) reverse transcription polymerase chain reaction (RT-PCR) detection of corneal tissue SP-A, SP-D mRNA expression; immunohistochemistry detection of corneal tissue SP-A, SP-D protein expression and co-expression intensity.
Results: (1) Slit lamp examination: There was no significant difference between the fungal infected cornea and the injured cornea at 6 h, but the conjunctiva was hyperemic (+) and the corneal edema was not obvious; the conjunctiva was hyperemic (++) at 12 h, the cornea was edematous; the conjunctiva was hyperemic (++++) at 24 h, and the cornea was ground glass edema at 48 h and 96 h; the corneal edema was obvious and the ulcer was yellow and white. (2) Histopathological examination: there was no significant difference between the fungal infected cornea and the simple injured cornea at 6 h. The central epithelial layer of cornea was absent, the collagen fibers were arranged neatly, and no obvious neutrophil infiltration was found; a small amount of neutrophil invasion was found in the superficial layer of stroma at 12 h; corneal edema at 24 h, and the central cornea was damaged. The loss of the skin and the anterior elastic layer, the disorder of collagen fibers in the stroma, the increase of neutrophil invasion in the superficial layer of the stroma, the obvious corneal edema at 48h and 96h, the disorder of corneal stroma arrangement, and the infiltration of a large number of neutrophils in the whole stroma. (3) Immunohistochemical detection: normal rats, simple injury group and fungal keratitis two groups of corneal epithelial cells. There was no significant difference in the expression of SP-A and SP-D proteins among the three groups (p0.05). The expression of SP-A and SP-D proteins in corneal epithelial cells of fungal infection cornea was not significantly increased at 6 hours, but there was no significant difference compared with normal cornea, simple injury cornea and fungal keratitis (p0.05). The expression gradually increased after 12 hours, the strongest at 48 hours, 96 hours. The expression level of SP-A mRNA and SP-D-mR in cornea of normal rats, simple injury group and fungal keratitis group were significantly different (p0.01). There was no significant difference in the expression of NA among the three groups (p0.05). The expression of SP-A mRNA and SP-D mRNA increased gradually after fungal infection, the highest at 24 hours, the lowest at 48 hours and the lowest at 96 hours. Differences (P0.01)
CONCLUSION: The expression of SP-A and SP-D in the cornea of Fusarium solani infection in early stage increased significantly, but decreased with the progression of the disease, but still higher than that in the simple injury group.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R772.21

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